Wild lime psyllid Leuronota fagarae Burckhardt (Hemiptera: Psylloidea) picorna-like virus full genome annotation and classification.

Picorna-like viruses of the order Picornavirales are a poorly defined group of positive-sense, single-stranded RNA viruses that include numerous pathogens known to infect plants, animals, and insects. A new picorna-like viral species was isolated from the wild lime psyllid (WLP), Leuronota fagarae, in the state of Florida, USA, and labelled: Leuronota fagarae picorna-like virus isolate FL (LfPLV-FL). The virus was found to have homology to a picorna-like virus identified in the Asian Citrus Psyllid (ACP), Diaphorina citri, collected in the state of Florida. Computational analysis of RNA extracts from WLP adult heads identified a 10,006-nucleotide sequence encoding a 2,942 amino acid polyprotein with similar functional domain structure to polyproteins of both Dicistroviridae and Iflaviridae. Sequence comparisons of nucleic acid and amino acid translations of the conserved RNA-dependent RNA polymerase, along with the entire N-terminal nonstructural coding region, provided insight into an evolutionary relationship of LfPLV-FL to insect-infecting iflaviruses. Viruses belonging to the family Iflaviridae encode a polyprotein of around 3000 amino acids in length that is processed post-translationally to produce components necessary for replication. The classification of a novel picorna-like virus in L. fagarae, with evolutionary characteristics similar to picorna-like viruses infecting Bactericera cockerelli and D. citri, provides an opportunity to examine virus host specificity, as well as identify critical components of the virus' genome required for successful transmission, infection, and replication. This bioinformatic classification allows for further insight into a novel virus species, and aids in the research of a closely related virus of the invasive psyllid, D. citri, a major pest of Floridian citriculture. The potential use of viral pathogens as expression vectors to manage the spread D. citri is an area that requires additional research; however, it may bring forth an effective control strategy to reduce the transmission of Candidatus Liberibacter asiaticus (CLas), the causative agent of Huanglongbing (HLB).

First Report of Lasiodiplodia pseudotheobromae causing Postharvest Decay of Strawberries in Florida.

Postharvest decay of strawberry (Fragaria × ananassa Duch.) is a major factor causing fruit losses. Strawberries were obtained from various harvests at cooling facilities located in Dover and Plant City, FL during the 2018-19 and 2019-20 seasons. After the fruits were incubated at 22ºC for up to 5 days (d) to promote disease development, Lasiodiplodia decay was observed at up to 3% from some harvests, exhibiting gray mycelia on small lesions that gradually covered the whole fruit. The fungus was isolated onto potato dextrose agar (PDA). Five isolates (SBD18-14, SBD18-277, SBD18-279, SBD19-02 and SBD19-57) were characterized. Fungal mycelia were initially grayish white and then gradually changed to gray to dark gray on PDA at 25oC, and later produced black pigments (Fig. S1). Pycnidia were observed from inoculated strawberries at 14 d. Isolates shared similar conidia morphology: aseptate, hyaline, ellipsoid to ovoid, measuring L × W: 24.0-34.0 (28.3) × 13.0-16.0 (14.3) µm (n =100). Mature conidia were brown, one septate, measuring L × W: 25.0-33.0 (28.8) × 13.0-16.0 (14.5) µm (n =100). The isolates were identified as Lasiodiplodia spp. morphologically (Alves et al. 2008). DNA was extracted from fungal mycelia using an OmniPrep DNA extraction kit, and PCR amplification of ITS and EF1-α genes was performed following the conditions described by White et al. (1990) with some modifications using primers ITS1F-F/ITS4-R (Gardes and Bruns, 1993; White et al., 1990) and EF1-668-F/EF1-1251-R (Alves et al., 2008), respectively. The BLASTn in GenBank showed that the sequences obtained had 99.61 to 100% homology with those of ITS (EF622077) and EF1-α (EF622057) from L. pseudotheobromae CBS116459 (an ex-type strain) (Alves et al., 2008). Sequences of the isolates have been deposited in GenBank with accessions OP326017 to OP326021 for ITS, and OP356202 to OP356206 for EF1-α. Phylogenetic analysis showed that these isolates clustered in the same clade (bootstrap value at 64) with L. pseudotheobromae (Fig. S2). Two fungal inoculum types (mycelia and conidia), two fruit inoculation methods (injury and non-injury) and five fungal isolates were used for pathogenicity tests. Fungal mycelia (2-day-old) on PDA plug (5 mm) or 10 µL of conidial suspension (106 spores/mL) was placed onto each injury (1 x 1 mm in size) or a non-injury area on the surfaces of five strawberry fruits (cv. Florida Brilliance). PDA plug alone or water drops placed on injury or non-injury areas on fruits served as respective controls. Inoculated and control fruits were incubated in a covered plastic container with 100% RH at 22ºC. The experiment was repeated twice. Decay initially appeared as soft and lightly discolored tissue at inoculation areas 2 d post-inoculation (dpi) that extended quickly thereafter. Brown to dark lesions on both injury- and non-injury fruits inoculated with conidia or mycelia were observed at 3 dpi. Decay and gray mycelia gradually developed over the whole fruit at 6 dpi, and pycnidia were observed after 14 dpi (Fig. S1). Disease incidence of 100% was observed on all tests. Control fruits did not develop decay. The results indicate that these isolates are pathogenic to strawberries and infect fruit via both non-injured and injured fruit surfaces. The inoculated fungal isolates were re-isolated, thus, fulfilling Koch's postulates. L. theobromae, Neofusicoccum parvum/N. ribis species complex causing strawberry fruit rot in Florida fields was reported (Oliveira et al., 2019), but not L. pseudotheobromae. To our knowledge, this is the first report of postharvest decay caused by L. pseudotheobromae A.J.L. Phillips, A. Alves & Crous on strawberries in Florida and in the USA, and it should be considered in strawberry disease management.

Draft Genome Sequence Resource for the Orange Rust Pathogen of Sugarcane Puccinia kuehnii.

Puccinia kuehnii is an obligate biotrophic fungal pathogen that causes orange rust of sugarcane, which is prevalent in many countries around the globe. In the United States, orange rust was first detected in sugarcane in Florida in 2007 and poses a persistent and economically damaging threat to the sugarcane industry in this region. Here, we generated the first genome assemblies for two isolates of P. kuehnii (1040 and 2143) collected in Florida in 2017 from two sugarcane cultivars, CL85-1040 and CP89-2143, respectively. These two rust genome resources will be of immense value for future genomic studies, particularly further exploration of the predicted secretomes that may help define key pathogenicity determinants for this economically important pathogen.

Draft Genome Sequence Resource of the Citrus Stem-End Rot Fungal Pathogen Lasiodiplodia theobromae CITRA15.

Lasiodiplodia theobromae is a fungal pathogen associated with perennial tropical fruit plants worldwide. In citrus, L. theobromae causes stem-end rot (Diplodia stem-end rot), a damaging postharvest disease that is aggravated when trees are also infected with the citrus greening bacteria 'Candidatus Liberibacter asiaticus'. Due to the latent infection of L. theobromae during the preharvest stage, it becomes difficult to control the disease by chemical or physical treatment. In the current study, we sequenced and assembled strain CITRA15, the first genome of L. theobromae obtained from diseased Citrus paradise 'Flame' grapefruit in Florida, and thereby provided a genomic resource for future research on diagnostics, and postharvest and preharvest disease management of citrus and other fruit crops.

Temporal and spatial detection of Candidatus Liberibacter asiaticus putative effector transcripts during interaction with Huanglongbing-susceptible, -tolerant, and -resistant citrus hosts.

BACKGROUND: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. RESULTS: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. CONCLUSION: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation.

Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation.

BACKGROUND: Oomycetes are a group of filamentous eukaryotic microorganisms that have colonized all terrestrial and oceanic ecosystems, and they include prominent plant pathogens. The Aphanomyces genus is unique in its ability to infect both plant and animal species, and as such exemplifies oomycete versatility in adapting to different hosts and environments. Dissecting the underpinnings of oomycete diversity provides insights into their specificity and pathogenic mechanisms. RESULTS: By carrying out genomic analyses of the plant pathogen A. euteiches and the crustacean pathogen A. astaci, we show that host specialization is correlated with specialized secretomes that are adapted to the deconstruction of the plant cell wall in A. euteiches and protein degradation in A. astaci. The A. euteiches genome is characterized by a large repertoire of small secreted protein (SSP)-encoding genes that are highly induced during plant infection, and are not detected in other oomycetes. Functional analysis revealed an SSP from A. euteiches containing a predicted nuclear-localization signal which shuttles to the plant nucleus and increases plant susceptibility to infection. CONCLUSION: Collectively, our results show that Aphanomyces host adaptation is associated with evolution of specialized secretomes and identify SSPs as a new class of putative oomycete effectors.

Evaluation of High-Resolution Melting for Rapid Differentiation of Phytophthora Hybrids and Their Parental Species.

Phytophthora species hybrids have been repeatedly reported as causing damaging diseases to cultivated and wild plants. Two known hybrids, P. andina and P. × pelgrandis, are pathogens of Solanaceae and ornamentals, respectively, although the extent of their host ranges are unknown. P. andina emerged from hybridization of P. infestans and an unidentified related species, whereas P. × pelgrandis emerged from P. nicotianae and P. cactorum. Considering that hybrids and parental species can coexist in the same regions and to distinguish them usually requires cloning or whole genome sequencing, we aimed to develop a rapid tool to distinguish them. Specifically, we used high-resolution melting (HRM) assays to differentiate genotypes based on their amplicon melting profiles. We designed primers for P. × pelgrandis and parental species based on available sequences of P. nicotianae and P. cactorum nuclear genes containing polymorphisms between species. For P. andina, heterozygous sites from Illumina short reads were used for the same purpose. We identified multiple amplicons exhibiting differences in melting curves between parental species and hybrids. We propose HRM as a rapid method for differentiation of P. andina and P. × pelgrandis hybrids from parental species that could be employed to advance research on these pathogens.

nQuire: a statistical framework for ploidy estimation using next generation sequencing.

BACKGROUND: Intraspecific variation in ploidy occurs in a wide range of species including pathogenic and nonpathogenic eukaryotes such as yeasts and oomycetes. Ploidy can be inferred indirectly - without measuring DNA content - from experiments using next-generation sequencing (NGS). We present nQuire, a statistical framework that distinguishes between diploids, triploids and tetraploids using NGS. The command-line tool models the distribution of base frequencies at variable sites using a Gaussian Mixture Model, and uses maximum likelihood to select the most plausible ploidy model. nQuire handles large genomes at high coverage efficiently and uses standard input file formats. RESULTS: We demonstrate the utility of nQuire analyzing individual samples of the pathogenic oomycete Phytophthora infestans and the Baker's yeast Saccharomyces cerevisiae. Using these organisms we show the dependence between reliability of the ploidy assignment and sequencing depth. Additionally, we employ normalized maximized log- likelihoods generated by nQuire to ascertain ploidy level in a population of samples with ploidy heterogeneity. Using these normalized values we cluster samples in three dimensions using multivariate Gaussian mixtures. The cluster assignments retrieved from a S. cerevisiae population recovered the true ploidy level in over 96% of samples. Finally, we show that nQuire can be used regionally to identify chromosomal aneuploidies. CONCLUSIONS: nQuire provides a statistical framework to study organisms with intraspecific variation in ploidy. nQuire is likely to be useful in epidemiological studies of pathogens, artificial selection experiments, and for historical or ancient samples where intact nuclei are not preserved. It is implemented as a stand-alone Linux command line tool in the C programming language and is available at https://github.com/clwgg/nQuire under the MIT license.

Gene expression polymorphism underpins evasion of host immunity in an asexual lineage of the Irish potato famine pathogen.

BACKGROUND: Outbreaks caused by asexual lineages of fungal and oomycete pathogens are a continuing threat to crops, wild animals and natural ecosystems (Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ, Nature 484:186-194, 2012; Kupferschmidt K, Science 337:636-638, 2012). However, the mechanisms underlying genome evolution and phenotypic plasticity in asexual eukaryotic microbes remain poorly understood (Seidl MF, Thomma BP, BioEssays 36:335-345, 2014). Ever since the 19th century Irish famine, the oomycete Phytophthora infestans has caused recurrent outbreaks on potato and tomato crops that have been primarily caused by the successive rise and migration of pandemic asexual lineages (Goodwin SB, Cohen BA, Fry WE, Proc Natl Acad Sci USA 91:11591-11595, 1994; Yoshida K, Burbano HA, Krause J, Thines M, Weigel D, Kamoun S, PLoS Pathog 10:e1004028, 2014; Yoshida K, Schuenemann VJ, Cano LM, Pais M, Mishra B, Sharma R, Lanz C, Martin FN, Kamoun S, Krause J, et al. eLife 2:e00731, 2013; Cooke DEL, Cano LM, Raffaele S, Bain RA, Cooke LR, Etherington GJ, Deahl KL, Farrer RA, Gilroy EM, Goss EM, et al. PLoS Pathog 8:e1002940, 2012). However, the dynamics of genome evolution within these clonal lineages have not been determined. The objective of this study was to use a comparative genomics and transcriptomics approach to determine the molecular mechanisms that underpin phenotypic variation within a clonal lineage of P. infestans. RESULTS: Here, we reveal patterns of genomic and gene expression variation within a P. infestans asexual lineage by comparing strains belonging to the South American EC-1 clone that has dominated Andean populations since the 1990s (Yoshida K, Burbano HA, Krause J, Thines M, Weigel D, Kamoun S, PLoS Pathog 10e1004028, 2014; Yoshida K, Schuenemann VJ, Cano LM, Pais M, Mishra B, Sharma R, Lanz C, Martin FN, Kamoun S, Krause J, et al. eLife 2:e00731, 2013; Delgado RA, Monteros-Altamirano AR, Li Y, Visser RGF, van der Lee TAJ, Vosman B, Plant Pathol 62:1081-1088, 2013; Forbes GA, Escobar XC, Ayala CC, Revelo J, Ordonez ME, Fry BA, Doucett K, Fry WE, Phytopathology 87:375-380, 1997; Oyarzun PJ, Pozo A, Ordonez ME, Doucett K, Forbes GA, Phytopathology 88:265-271, 1998). We detected numerous examples of structural variation, nucleotide polymorphisms and loss of heterozygosity within the EC-1 clone. Remarkably, 17 genes are not expressed in one of the two EC-1 isolates despite apparent absence of sequence polymorphisms. Among these, silencing of an effector gene was associated with evasion of disease resistance conferred by a potato immune receptor. CONCLUSIONS: Our findings highlight the molecular changes underpinning the exceptional genetic and phenotypic plasticity associated with host adaptation in a pandemic clonal lineage of a eukaryotic plant pathogen. We observed that the asexual P. infestans lineage EC-1 can exhibit phenotypic plasticity in the absence of apparent genetic mutations resulting in virulence on a potato carrying the Rpi-vnt1.1 gene. Such variant alleles may be epialleles that arose through epigenetic changes in the underlying genes.

Arabidopsis late blight: infection of a nonhost plant by Albugo laibachii enables full colonization by Phytophthora infestans.

The oomycete pathogen Phytophthora infestans causes potato late blight, and as a potato and tomato specialist pathogen, is seemingly poorly adapted to infect plants outside the Solanaceae. Here, we report the unexpected finding that P. infestans can infect Arabidopsis thaliana when another oomycete pathogen, Albugo laibachii, has colonized the host plant. The behaviour and speed of P. infestans infection in Arabidopsis pre-infected with A. laibachii resemble P. infestans infection of susceptible potato plants. Transcriptional profiling of P. infestans genes during infection revealed a significant overlap in the sets of secreted-protein genes that are induced in P. infestans upon colonization of potato and susceptible Arabidopsis, suggesting major similarities in P. infestans gene expression dynamics on the two plant species. Furthermore, we found haustoria of A. laibachii and P. infestans within the same Arabidopsis cells. This Arabidopsis-A. laibachii-P. infestans tripartite interaction opens up various possibilities to dissect the molecular mechanisms of P. infestans infection and the processes occurring in co-infected Arabidopsis cells.

Rerouting of plant late endocytic trafficking toward a pathogen interface.

A number of plant pathogenic and symbiotic microbes produce specialized cellular structures that invade host cells where they remain enveloped by host-derived membranes. The mechanisms underlying the biogenesis and functions of host-microbe interfaces are poorly understood. Here, we show that plant late endocytic trafficking is diverted toward the extrahaustorial membrane (EHM); a host-pathogen interface that develops in plant cells invaded by Irish potato famine pathogen Phytophthora infestans. A late endosome and tonoplast marker protein Rab7 GTPase RabG3c, but not a tonoplast-localized sucrose transporter, is recruited to the EHM, suggesting specific rerouting of vacuole-targeted late endosomes to a host-pathogen interface. We revealed the dynamic nature of this process by showing that, upon activation, a cell surface immune receptor traffics toward the haustorial interface. Our work provides insight into the biogenesis of the EHM and reveals dynamic processes that recruit membrane compartments and immune receptors to this host-pathogen interface.

Host specialization of the blast fungus Magnaporthe oryzae is associated with dynamic gain and loss of genes linked to transposable elements.

BACKGROUND: Magnaporthe oryzae (anamorph Pyricularia oryzae) is the causal agent of blast disease of Poaceae crops and their wild relatives. To understand the genetic mechanisms that drive host specialization of M. oryzae, we carried out whole genome resequencing of four M. oryzae isolates from rice (Oryza sativa), one from foxtail millet (Setaria italica), three from wild foxtail millet S. viridis, and one isolate each from finger millet (Eleusine coracana), wheat (Triticum aestivum) and oat (Avena sativa), in addition to an isolate of a sister species M. grisea, that infects the wild grass Digitaria sanguinalis. RESULTS: Whole genome sequence comparison confirmed that M. oryzae Oryza and Setaria isolates form a monophyletic and close to another monophyletic group consisting of isolates from Triticum and Avena. This supports previous phylogenetic analysis based on a small number of genes and molecular markers. When comparing the host specific subgroups, 1.2-3.5 % of genes showed presence/absence polymorphisms and 0-6.5 % showed an excess of non-synonymous substitutions. Most of these genes encoded proteins whose functional domains are present in multiple copies in each genome. Therefore, the deleterious effects of these mutations could potentially be compensated by functional redundancy. Unlike the accumulation of nonsynonymous nucleotide substitutions, gene loss appeared to be independent of divergence time. Interestingly, the loss and gain of genes in pathogens from the Oryza and Setaria infecting lineages occurred more frequently when compared to those infecting Triticum and Avena even though the genetic distance between Oryza and Setaria lineages was smaller than that between Triticum and Avena lineages. In addition, genes showing gain/loss and nucleotide polymorphisms are linked to transposable elements highlighting the relationship between genome position and gene evolution in this pathogen species. CONCLUSION: Our comparative genomics analyses of host-specific M. oryzae isolates revealed gain and loss of genes as a major evolutionary mechanism driving specialization to Oryza and Setaria. Transposable elements appear to facilitate gene evolution possibly by enhancing chromosomal rearrangements and other forms of genetic variation.

Host-induced aneuploidy and phenotypic diversification in the Sudden Oak Death pathogen Phytophthora ramorum.

BACKGROUND: Aneuploidy can result in significant phenotypic changes, which can sometimes be selectively advantageous. For example, aneuploidy confers resistance to antifungal drugs in human pathogenic fungi. Aneuploidy has also been observed in invasive fungal and oomycete plant pathogens in the field. Environments conducive to the generation of aneuploids, the underlying genetic mechanisms, and the contribution of aneuploidy to invasiveness are underexplored. We studied phenotypic diversification and associated genome changes in Phytophthora ramorum, a highly destructive oomycete pathogen with a wide host-range that causes Sudden Oak Death in western North America and Sudden Larch Death in the UK. Introduced populations of the pathogen are exclusively clonal. In California, oak (Quercus spp.) isolates obtained from trunk cankers frequently exhibit host-dependent, atypical phenotypes called non-wild type (nwt), apparently without any host-associated population differentiation. Based on a large survey of genotypes from different hosts, we previously hypothesized that the environment in oak cankers may be responsible for the observed phenotypic diversification in P. ramorum. RESULTS: We show that both normal wild type (wt) and nwt phenotypes were obtained when wt P. ramorum isolates from the foliar host California bay (Umbellularia californica) were re-isolated from cankers of artificially-inoculated canyon live oak (Q. chrysolepis). We also found comparable nwt phenotypes in P. ramorum isolates from a bark canker of Lawson cypress (Chamaecyparis lawsoniana) in the UK; previously nwt was not known to occur in this pathogen population. High-throughput sequencing-based analyses identified major genomic alterations including partial aneuploidy and copy-neutral loss of heterozygosity predominantly in nwt isolates. Chromosomal breakpoints were located at or near transposons. CONCLUSION: This work demonstrates that major genome alterations of a pathogen can be induced by its host species. This is an undocumented type of plant-microbe interaction, and its contribution to pathogen evolution is yet to be investigated, but one of the potential collateral effects of nwt phenotypes may be host survival.

A Recent Expansion of the RXLR Effector Gene Avrblb2 Is Maintained in Global Populations of Phytophthora infestans Indicating Different Contributions to Virulence.

The introgression of disease resistance (R) genes encoding immunoreceptors with broad-spectrum recognition into cultivated potato appears to be the most promising approach to achieve sustainable management of late blight caused by the oomycete pathogen Phytophthora infestans. Rpi-blb2 from Solanum bulbocastanum shows great potential for use in agriculture based on preliminary potato disease trials. Rpi-blb2 confers immunity by recognizing the P. infestans avirulence effector protein AVRblb2 after it is translocated inside the plant cell. This effector belongs to the RXLR class of effectors and is under strong positive selection. Structure-function analyses revealed a key polymorphic amino acid (position 69) in AVRblb2 effector that is critical for activation of Rpi-blb2. In this study, we reconstructed the evolutionary history of the Avrblb2 gene family and further characterized its genetic structure in worldwide populations. Our data indicate that Avrblb2 evolved as a single-copy gene in a putative ancestral species of P. infestans and has recently expanded in the Phytophthora spp. that infect solanaceous hosts. As a consequence, at least four variants of AVRblb2 arose in P. infestans. One of these variants, with a Phe residue at position 69, evades recognition by the cognate resistance gene. Surprisingly, all Avrblb2 variants are maintained in pathogen populations. This suggests a potential benefit for the pathogen in preserving duplicated versions of AVRblb2, possibly because the variants may have different contributions to pathogen fitness in a diversified solanaceous host environment.

Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora.

BACKGROUND: Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily. RESULTS: Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic. CONCLUSIONS: The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations.

The majority of agronomically important crop traits are quantitative, meaning that they are controlled by multiple genes each with a small effect (quantitative trait loci, QTLs). Mapping and isolation of QTLs is important for efficient crop breeding by marker-assisted selection (MAS) and for a better understanding of the molecular mechanisms underlying the traits. However, since it requires the development and selection of DNA markers for linkage analysis, QTL analysis has been time-consuming and labor-intensive. Here we report the rapid identification of plant QTLs by whole-genome resequencing of DNAs from two populations each composed of 20-50 individuals showing extreme opposite trait values for a given phenotype in a segregating progeny. We propose to name this approach QTL-seq as applied to plant species. We applied QTL-seq to rice recombinant inbred lines and F2 populations and successfully identified QTLs for important agronomic traits, such as partial resistance to the fungal rice blast disease and seedling vigor. Simulation study showed that QTL-seq is able to detect QTLs over wide ranges of experimental variables, and the method can be generally applied in population genomics studies to rapidly identify genomic regions that underwent artificial or natural selective sweeps.

Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen.

Pest and pathogen losses jeopardise global food security and ever since the 19(th) century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.

Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface.

In response to pathogen attack, plant cells secrete antimicrobial molecules at the site of infection. However, how plant pathogens interfere with defense-related focal secretion remains poorly known. Here we show that the host-translocated RXLR-type effector protein AVRblb2 of the Irish potato famine pathogen Phytophthora infestans focally accumulates around haustoria, specialized infection structures that form inside plant cells, and promotes virulence by interfering with the execution of host defenses. AVRblb2 significantly enhances susceptibility of host plants to P. infestans by targeting the host papain-like cysteine protease C14 and specifically preventing its secretion into the apoplast. Plants altered in C14 expression were significantly affected in susceptibility to P. infestans in a manner consistent with a positive role of C14 in plant immunity. Our findings point to a unique counterdefense strategy that plant pathogens use to neutralize secreted host defense proteases. Effectors, such as AVRblb2, can be used as molecular probes to dissect focal immune responses at pathogen penetration sites.

MutMap-Gap: whole-genome resequencing of mutant F2 progeny bulk combined with de novo assembly of gap regions identifies the rice blast resistance gene Pii.

Next-generation sequencing allows the identification of mutations responsible for mutant phenotypes by whole-genome resequencing and alignment to a reference genome. However, when the resequenced cultivar/line displays significant structural variation from the reference genome, mutations in the genome regions missing from the reference (gaps) cannot be identified by simple alignment. Here we report on a method called 'MutMap-Gap', which involves delineating a candidate region harboring a mutation of interest using the recently reported MutMap method, followed by de novo assembly, alignment, and identification of the mutation within genome gaps. We applied MutMap-Gap to isolate the blast resistant gene Pii from the rice cv Hitomebore using mutant lines that have lost Pii function. MutMap-Gap should prove useful for cloning genes that exhibit significant structural variations such as disease resistance genes of the nucleotide-binding site-leucine rich repeat (NBS-LRR) class.